The present invention relates to a method for detecting pathological and metabolic changes related to alcohol intake in an individual, and more particularly to a method for detecting alcohol intake and alcohol related liver damage by measuring the sialylation index of apolipoprotein J.
There are about 10 million alcoholics in the United States. Alcoholic liver disease is a major cause of morbidity and mortality in the United States and in many other countries in the world. Ethanol is toxic to the liver. Thus, the persistent, excessive intake of ethanol can lead to a number of complications in the human body, including damage to liver cells and eventually, to cirrhosis of the liver.
Profound changes in the concentration and composition of plasma lipids and lipoproteins occur at each stage of excessive alcohol intake and/or alcohol-induced liver injury. Ghosh, P. et al., xe2x80x9cLong-Term Ethanol Exposure Impairs Glycosylation of Both Nxe2x80x94 and Oxe2x80x94 Glycosylated Proteins in Rat Liver,xe2x80x9d Metabolism 44: 890-898 (1995). One of the main sites of ethanol attack is the glycosylation machinery of the liver. This deleterious action of ethanol manifests itself by altering specific activities of the sialylation and desialylation enzymes of the liver which results in either defective sialylation of proteins or increased hydrolysis of molecules that eventually result in the depletion of sialic acid residues from sialylomolecules. See Ghosh, P. et al., xe2x80x9cEffects of Chronic Ethanol on Enzymes Regulating Sialylation and Desialylation of Transferrin in Rats,xe2x80x9d Alcoh. Clin. Exp. Res. 17: 576-579 (1993). Hepatocellular degeneration results in a variety of clinical symptoms ranging from a relatively asymptomatic enlargement of the liver to massive fatty infiltration. As the alcohol abuse continues, these degenerative processes manifest themselves into liver dysfunction, chronic inflammation, and structural distortion of cells leading to proliferation of fibrous tissue, and ultimately to cirrhosis and necrosis of the liver. Total hepatic failure and death may occur as a result of prolonged alcohol abuse in humans.
Self-reporting of alcohol intake is unreliable. Accordingly, tests to diagnose alcohol intake have been developed. Serum carbohydrate-deficient transferrin (CDT) is considered to be a viable marker for alcohol intake. Transferrin is a sialoglycoprotein that transports iron into cells and is present at high concentrations in serum. Excessive alcohol intake by an individual, or ingestion of greater than 60 grams of ethanol daily, produces high levels of CDT in serum. Normal individuals and non-alcoholic liver disease patients do not exhibit the elevation of CDT (except in a few primary biliary cirrhosis or hepatitis).
Tests based on the CDT marker are generally directed to identifying and quantifying CDT in a sample. U.S. Pat. No. 4,626,355 discloses a method for determining the alcohol intake in an individual by quantifying the isotransferrins (CDT) in the individual""s body fluids. The method taught in U.S. Pat. No. 5,432,059 entails detecting CDT by reglycosylating (with a fluorescent-conjugate) deglycosylated glycoproteins in a sample; detecting the fluorescent, reglycosylated glycoprotein using a flurometer; and quantifying the amount of reglycosylated glycoproteins in a sample. U.S. Pat. No. 5,702,904 discloses a CDT based immunoassay which utilizes an antibody that reacts specifically with CDT.
The CDT tests are laborious, time consuming and expensive. More importantly, the validity of the CDT marker has been seriously questioned. In fact, the CDT marker has been reported to be invalid in alcoholics who consume less that 60 grams of alcohol per day, pregnant women, and in patients with hepatitis. See Stowell, L. I. et al., xe2x80x9cCDT test is not valid in detecting less excessive regular drinking in young males and females,xe2x80x9dAlcohol Alcohol 32: 507-517 (1997); Lesch, O.N. et al., xe2x80x9cNo correlation of CDT with alcohol-related disabilities, severity of withdrawal syndrome,xe2x80x9d Alcohol Alcohol 31: 257-264 (1996); Lesch, O. N. et al., xe2x80x9cCDT not sensitive for short-term heavy drinking by healthy subjects,xe2x80x9d Alcohol Alcohol 31: 265-271 (1996). The result of a recent study conducted jointly by NIAAA, NHLBI and VA, showed that CDT levels were unaffected by as large as a 50% decrease in the amount of alcohol consumed by the study subjects. See Lakshman, R., A report submitted to the NIH by Study Director Raj Lakshman, Ph.D. (personal communication). Therefore, a cost-effective and reliable marker for alcohol intake and alcohol related liver damage is still needed for early diagnosis and treatment of alcoholism and related liver damage.
One aspect of the present invention is directed to a method for detecting alcohol intake and alcohol related liver damage in an individual, which involves the following steps:
a. providing a sample containing Apolipoprotein J (Apo J);
b. purifying the Apo J;
c. determining sialic acid content of the purified Apo J relative to protein content of Apo J in the sample; and
d. evaluating whether the sialylation acid index of Apo J is an indication of alcohol intake or alcohol related liver damage in the subject, or the extent of detoxification or recovery from alcohol related illness.
In preferred embodiments, the Apo J protein is purified via immunoaff inity chromatography matrix, such as a column comprising immobilized antibodies that specifically bind Apo J. The Apo J sample is obtained in blood, serum, plasma or high density lipoprotein (HDL). The sialic acid content is determined by subjecting the purified Apo J to acid hydrolysis to release the sialic acid residues from the apolipoprotein. The content of sialic acid is expressed in terms of a sialylation index (SI), i.e., moles of sialic acid per mole of Apo J protein. The SI ratio obtained is compared to a control or standard SI generated from non-drinkers as an indication of alcohol intake and/or liver damage.
Another aspect of this invention is directed to a kit for measuring the sialic acid content of Apo J. The kit contains a matrix having immobilized anti-Apo J antibodies and a desialylation agent. In preferred embodiments, the matrix is contained in a column, and the desialylation agent is an enzyme or acid such as sulfuric acid. In another preferred embodiment, the kit also contains a desalting column and washing and elution buffers.
Apo J is a 70 kilodalton apolipoprotein component of plasma high density lipoproteins. Applicant has discovered that the Apo J sialic acid test is more sensitive than the CDT test to changes in alcohol intake. In a side-by-side comparison, Applicant demonstrated that preferred forms of the present invention can provide as much as about 20% more sensitivity than tests based on the CDT marker. The present invention offers a better alternative to the CDT test because it is specific, sensitive, simple, and cost-effective.